Entomological survey of the potential vectors of Rift Valley fever virus and absence of detection of the virus genome from the vectors in various niches in the southern half of the Great Rift Valley of Ethiopia

Abstract Background Rift Valley fever virus (RVFV) is the cause of one of the most important mosquito‐borne emerging diseases negatively affecting the health of humans and animals, particularly in Africa. In Ethiopia, the status of RVFV and the existence of potential vectors are unknown. Objectives This study aimed to survey the mosquito vectors of RVFV and the detection of the virus in selected sites (Batu, Hawassa, Arba Minch and Borana) in Ethiopia. Methods CDC light traps baited with the sugar‐yeast solution were set up at various locations for a total of 29 trap nights. Mosquitoes identification were made morphologically using a stereomicroscope and for RVFV detection by reverse transcriptase‐polymerase chain reaction (RT‐PCR). Results Among a total of 132 trap efforts conducted, 60 (45%) captured the mosquitoes. A total of 1576 adult mosquitoes were collected and identified. Including Aedes (n = 407; 25.8%), Anopheles (n = 493; 32.3%), Culex (n = 466; 29.6%) and Mansonia (n = 210; 13.32%). The genome material of RVFV was not detected by RT‐PCR. Conclusions The existence of a potential Aedes species may pose a risk for the occurrence of the RVF outbreak in Ethiopia. Based on the current study, we recommend further monitoring for potential mosquito vectors of RVFV, particularly with a view to targeting the seasons during which the mosquitoes can be abundant along with a serological survey of susceptible hosts.


INTRODUCTION
Mosquito-borne viral diseases are becoming a major public and animal health challenge (Cooper et al., 2015;Wilder-Smith et al., 2017).
Rift Valley fever (RVF) is a mosquito-borne viral disease with veterinary and public health implications (Pepin et al., 2010;Nanyingi et al., 2015). The causative agent of the disease is the RVF virus (RVFV) of the genus Phlebovirus, family Phenuiviridae, order Bunyavirales (Abudurexiti et al., 2019). Historical perspective showed that the virus was first identified in 1930, when it caused an outbreak of sudden mass deaths and abortions in sheep around Lake Naivasha in the Greater Rift Valley of Kenya (Daubney et al., 1931). Subsequently, outbreaks of RVF have been reported in some other African countries (Davies, 2010) such as Egypt in 1977, Madagascar in 1979and Mauritania in 1989(Pepin et al., 2010 and more recently in 2000 in Middle East countries such as Saudi Arabia and Yemen (Miller et al., 2002). In general, RVF is widespread in Africa where 36 African countries have reported cases of the disease (Leta et al., 2018).
RVFV has a wide range of natural hosts, of which the virus infection was reported from domestic ruminants and camels (Wright et al., 2019;Tchouassi et al., 2016), many wildlife (Rostal et al., 2017), and humans (LaBeaud et al., 2008). There are differences in the susceptibility to the virus in domestic animals with sheep being highly susceptible (Mansfield et al., 2015). RVFV is adapted to a variety of mosquitoes (Arum et al., 2015), and mammals usually acquire the viral infection through mosquito (Diptera: Culicidae) bites. In regions where RVF epidemics occurred, the virus has been isolated from at least 53 mosquito species which are grouped into eight genera .
The mosquito vectors that transmit RVFV are classified into two major groups, namely primary and secondary vectors (Arum et al., 2015).
Some mosquitoes of the genus Aedes (also called floodwater mosquito) are considered to be the primary maintenance host and source of RVFV that initiates disease outbreaks (Himeidan et al., 2014;Arum et al., 2015;Sang et al., 2017). The genera Culex, Eretmopodites and Mansonia constitute the secondary vectors with an affinity for flooded grounds to lay their eggs. Mosquitoes in this group, due to their ubiquitous bite patterns, contribute to the amplification of the virus, which subsequently leads to disease outbreaks (USG agencies Working Group, 2015;Arum et al., 2015). RVFV is also mechanically transmitted by other arthropods such as Culicoides, sand flies and Stomoxys calcitrans (Hoch et al., 1985;Turell et al., 2010).
The epidemiology of RVFV is highly dynamic and intricate under which effective transmission mainly depends on various factors such as the availability of competent vectors, susceptible hosts, and suitable ecological and environmental conditions that support mosquito survival and reproduction (Chevalier, 2013;Iaconoa et al., 2018;Arum et al., 2015). Once the virus is brought into favourable ecologies, it becomes endemic with periodic outbreaks and can spread even further into non-endemic environments of permissive areas (Baba et al., 2016).
RVF has notable socioeconomic and public health impacts associated with high mortality and morbidity in livestock and human with subsequent negative economic impact through livestock trade ban (Muga et al., 2015). Countries in the Horn of Africa have been affected by RVF. Kenya in particular has experienced multiple outbreaks, with about 23 outbreaks reported between 1912 to 2007 that resulted in several human fatalities and the loss of large numbers of domestic ruminants (Baba et al., 2016;Mosomtai et al., 2016Mosomtai et al., , 2018 (Baba et al., 2016).
Although Ethiopia shares long borders with disease-endemic countries such as Kenya, Somalia and Sudan (Tran et al., 2016), there have been no reports of cases of RVF from Ethiopia except for some recent serological evidence (Ibrahim et al., 2021;Endale et al., 2021;Asebe et al., 2020).
According to a forecast model developed by Weledekidane (2018) using localised seasonal precipitation data, the Horn of Africa precipi- Considering the huge health and economic impacts of RVF as well as the high potential of the expansion of the disease to nonendemic areas, regular surveillance and prediction are important.
Specifically, the implementation of regular surveillance can greatly contribute towards designing an effective mechanism of the control and prevention of the disease (Wilson et al., 2014;Chevalier, 2013). Surveillance of the diseases, particularly in the part of the country shared borders with the neighbour endemic countries where information about the disease is insufficient or absent is particularly useful for control (Weledekidane, 2018;Tran et al., 2016). Similar to other mosquito-borne diseases, identification of potential mosquito species transmitting RVF is an important step that can help to acquire biological information such as oviposition sites, biting and resting habits that differ among mosquito species (Taira et al., 2012).
Some entomological surveys were conducted after the outbreak of other mosquito-borne viral diseases in Ethiopia. For example, during the yellow fever outbreak in the South Omo Zone from November 2012 to October 2013, a vector survey was conducted and Aedes aegypti was the most abundant among the mosquitoes collected (Lilay et al., 2017). Similarly, after dengue outbreaks in Dire Dawa, eastern Ethiopia (Getachew et al., 2015) and Metema and Humera, north Ethiopia (Ferede et al., 2018), mosquito larvae were collected and morphologically identified. However, an entomological study regarding the presence and distribution of potential vectors of RVFV has not yet been investigated. Therefore, this study aimed to investigate the potential mosquito vector of RVFV in different geographic niches and also to perform the molecular detection of the virus in the mosquitoes.

MATERIALS AND METHODS
This study focused on an entomological survey of mosquito vectors potentially transmitting RVFV at seven different specific sites in Ethiopia. In particular, the sites included in the present study can be grouped into two: (1) those sites supposed to favour the introduction (e.g., due to geographic proximity to the endemic areas) and (2) those locations considered to be significant maintenance for the vectors of RVFV (e.g., near bodies of water). The mosquitoes collected from different sites using CDC traps were also tested for the presence of the RVFV using RT-PCR.

Mosquito collection areas
This study was designed to investigate the mosquito vectors of RVFV  sugar-yeast solution. The traps were placed near potential mosquito oviposition and feeding sites, including indoor and outdoor areas, particularly near the water bodies, near the animal enclosure, and in the field where dense human and livestock populations existed.

Mosquito collection and identification
Mosquitoes have diverse feeding behaviours, with some members of the genus Aedes being diurnal (biting mainly in the morning or evening) while others are nocturnal, while most members of the genera Anopheles, Culex and Mansonia are nocturnal feeders (Rozendaal, 1997). To accommodate the different feeding times of the mosquitoes, some of the traps were placed at 18:00 and collected between 6:00 and 7:00 h on the next day while others were set at around 16:00 and collected at 9:00 (the next day). The collection cups were deep frozen (-20 • C) for 15 min to tranquillise the mosquitoes for preservation. In the laboratory, sorting and identification of the genus and species levels was performed using dichotomous keys from the Walter Reed BioSystemics Unit (WRBU) (Potter, 2016) and Edwards (Edwards, 1941).
The morphology of the mosquitoes was carefully observed by an entomologist using a stereomicroscope. Some of the mosquitoes iden-

Viral genome extraction and amplification
At NVI's laboratory of, the preserved mosquito pools were processed to identify the presence of the RVFV genome in mosquitoes. For the purpose of extraction, each pool was grounded using a sterile mortar and pestle by adding 2-3 ml of phosphate buffered saline (PBS) containing antibiotics (penicillin, streptomycin and gentamycin). After centrifugation at 12,000 rpm for 10 min, the supernatant was aliquoted

Mosquito survey
Out of a total of 132 traps placed, it was possible to catch mosquitoes in 60 traps at different locations (Table 1) Mansonia (n = 210; 13.3%) and Anopheles (n = 493; 31 .3%). There were many mosquito body parts in trap cups that could not be identified because they were severely damaged and morphologically distorted.
The number of mosquitoes collected around water bodies was 1099 (69.73%), higher than other places as shown in Table 3. Of the mosquitoes identified, a total of 797 belonged to four genera, Aedes

Viral detection
Of 32 mosquito pools with 20-25 female mosquitoes per pool processed for viral nucleic acid detection by RT-PCR, none of the pools were positive for the RVFV genome.

DISCUSSION
The current entomological survey was conducted to assess the occur- Aedes mosquitoes were incriminated as principal vectors for RVFV and believed to play a significant role in maintaining the endemicity of the disease in the environment through transovarian transmission (Alhaj et al., 2017 occurrence of these two species and have been implicated as a reason for the initiation and spread of RVFV during disease outbreaks (Arum et al., 2015;Ochieng et al., 2016;Sang et al., 2017). Mosquitoes of the genus Culex have also been considered as potential vectors as a due to their bioecology in terms of abundance, biting activity, feeding habits and longevity (Brustolin et al., 2017). RVFV has been detected in many species of these mosquitoes in Madagascar (Tantely et al., 2015). In the present study, five Culex spp. (Table 2) have been identified and if the disease breaks out at the sites, there is a possibility that the disease will spread widely. Similarly, two Anopheles species (Anopheles arabiansis and Anopheles gambiae) were identified in the current study. These Anopheles species have been found to be infected with RVFV in Sudan (Seufi and Galal, 2010) and Kenya (Sang et al., 2017).
Mosquitoes typically search areas near bodies water to lay eggs (Che et al., 2013) and therefore, the occurrence and abundance of RVFV mosquito vectors are influenced by the type of biotope (temporary ponds, river or lakes) (Biteye et al., 2018). In general, Borana is an arid and semi-arid environment with minimal rainfall. A pond is the common body of water in the area, which is surface water harvested from rain in valleys. In Borana, a community pond is made by blocking floods in valleys when it rain or constructing a dam along a dry river during the dry season to collect the upcoming rainwater during the rainy season (Godana and Derib, 2021). During the current study period, these ponds have been reduced to less than their full size (full dam  (Sang et al., 2017). Others such as Cx. pipiens, Cx. antennatus and Ae. mcintoshi, which are competent vector of RVFV (Turell et al., 2008), have also been identified in the catches from Borana. The presence of potential vectors in this area, coupled with the nature of transboundary livestock movement (Lasage et al., 2010) and proximity to an endemic area, particularly Marsabit County in Kenya (Hassan et al., 2020), indicated the possibility of RVF outbreak could happen in the border area with Kenya.
The present study did not detect RVFV in the tested mosquitoes.
However, there are several limitations that need to be considered, so the results should be interpreted cautiously. Unfortunately, research funding was limited and this forced us to conduct most of the study near bodies of water during the dry season rather than during the more appropriate wet season. This resulted in only a small sample size of mosquitoes being collected and potential vectors may not have been captured and identified. Also, in the current study, we used a yeast-sugar solution as a CO 2 source to attract mosquitoes. This was because we could not sustain dry ice in field situations. Although yeastgenerated CO 2 is a convenient carbon dioxide source for mosquito trapping (Smallegange et al., 2010;Jerry et al., 2017), it is not as standardised as commercial CO 2 ; For example, the flow rate of CO 2 and the effects of other gaseous products are unknown and may have influenced mosquito collection.
The virus could circulate in the sub-detection level and spread if favourable conditions are met (Pepin et al., 2010 (Lichoti et al., 2014;Ibrahim et al., 2021;Lumley et al., 2018). Given the presence of potential vectors and circulating antibodies in Ethiopia, the possibility of an RVF outbreak cannot be excluded.
RVFV is transmitted to animals by the bites of infected mosquitoes, particularly those of the genera Aedes, Culex and Mansonia (Che et al., 2013). In contrast to the current absence of viral genome in mosquitoes, RVFV was detected from many similar mosquito species during the outbreak in Madagascar (Ratovonjato et al., 2011), Sudan and Egypt (Seufi and Galal, 2010), Kenya (Linthicum et al., 1985) and in experimental infection in Europe (Brustolin et al., 2017). However, during the inter-epidemic period, the probability of detecting the RVFV genome in mosquitoes is extremely low (Mhina et al., 2015;Pachka et al., 2016;Alhaj et al., 2017).
In conclusion, although no clinical cases of RVF have yet been observed and detection of the virus has not been reported in Ethiopia, (1) the presence of diversified competent RVFV mosquito vectors, (2) the country's geographical proximity to RVF-endemic countries and (3) the nature of livestock movements across the international border may lead to the conclusion that Ethiopia is at risk of RVF outbreak during epidemic periods in the Horn of Africa. Our findings provided up-to-date information that will help to set up efficient entomological and RVF disease surveillance and intervention strategies during high mosquito activity, particularly along border areas with endemic countries.